Cm1806

A half inch of terminal small intestine was separated for IHC. The intestine was gently washed with room temperature phosphate buffered saline (PBS), incubated for a minute in Tissue Plus O.C.T. Compound (Fisher HealthCare) and then flash-frozen in dry ice and 2-methylbutane and stored at −80°C until processing. Using a cryostat (Leica, Wetzlar, Germany, CM1806), samples were sectioned 5 μm thick at −20°C. Sections were fixed in −20°C methanol for 30 s. Non-specific binding of primary antibodies was blocked using 5% BSA for one hour at room temperature. Cells were then washed in room temperature PBS, 3 times for five min each and incubated with B0AT1 primary antibodies (Abcam 180516) at a 1:500 dilution for one hour. Cells were washed as before, then incubated with goat anti-rabbit Alexa Fluor 594 secondary antibodies (Invitrogen Life Technologies, Carlsbad, CA, USA) for one hour at a 1:500 dilution. The tissue sections were washed again, mounted with Fluoroshield Mounting Medium with 2′,6-diamidino-2-phenylindole (DAPI) (Abcam), imaged on an EVOS FL Auto 2 microscope (Invitrogen Life Technologies, Carlsbad, CA, USA) using a 20× objective and quantified with AlphaView software version 3.4.0.0.

SD rats were anesthetized with 2% pentobarbital sodium (40 mg/kg; Sigma-Aldrich) and transcardially perfused with 200 mL of 0.9% NaCl followed by 4% paraformaldehyde (PFA). Their brains were prepared and fixed in 4% PFA for 24 h, then they were stored at −80°C after gradient dehydration. The collected tissues were embedded in Tissue-Tek OCT compound (Sakura Finetek, Umkirch, Germany) and cut into 15-μm-thick sections with a freezing microtome (CM1806, Leica, Zurich, Switzerland); then they were incubated with the following antibodies: Mouse anti-GFAP antibody (1: 200, Proteintech Group, Chicago, USA), Goat Anti-Mouse IgG H&L (1: 400, Bioss, Beijing, China), Goat Anti-Mouse IgG H&L/Cy3 (1: 400, Bioss). Images were acquired using a confocal laser scanning microscope (Zeiss LSM700, Oberkochen, Germany) and quantified using Image J software. The quantity of GFAP immunoreactivity was expressed as GFAP-positive area in percentage of the total area in which GFAP-labelled immunoreactivity was determined.